Field of the Invention
The present invention relates to the fields of blood coagulation and immunology. More specifically, the present invention discloses peptide sequences that inhibit soluble fibrin (sFn) binding to blood monocytes and melanoma cells and their use in a wide variety of diseases such as cancer, cardiovascular disease, arthritis and inflammation.
Description of the Related Art
A relationship between cancer and abnormalities of the coagulation system has been recognized for over 100 years. Thromboembolic disease (usually of unknown etiology), refractory to anticoagulant therapy, may be an early detectable sign of an underlying cancer, which could precede the onset of observable cancer by months or years. Although many cancer patients exhibit clinically significant hemostatic abnormalities, about 50% of all patients (>90% with metastasis) have abnormal laboratory coagulation parameters (Gouin-Thibault & Samama., 1999). The most commonly reported abnormalities are elevated fibrinogen, fibrinopeptide A (FPA), raised platelet count and prolonged prothrombin time. Several studies have also reported increased soluble fibrin monomer (Iversen et al., 1995; Nakagawa et al, 1994; Andrassy et al., 1980). Patients with disseminated intravascular coagulation (DIC) have increased levels of circulating soluble fibrin monomer (Rickles et al., 1992). Persistently elevated levels of prothrombin fragment 1.2, thrombin-anti-thrombin complexes and soluble fibrin monomer, far above those seen in most thrombophilias may suggest an undiagnosed malignancy (Duncan et al., 1997). The presence of soluble fibrin in blood has, until recently, been considered a benign marker of the presence of an ongoing coagulopathy.
Soluble Fibrin
Fibrinogen is a homo-dimer of three peptide chains, designated Aa, Bb, and g. The protease thrombin binds to the Bb chain and cleaves the A peptide, followed by the B peptide from fibrinogen, leaving a fibrin monomer subunit. At sufficient concentration (as in clot formation) fibrin monomers will spontaneously polymerize to form insoluble fibrin polymer (the clot). However, during the initial stages of disseminated intravascular coagulation (induced by cancer, inflammation, sepsis etc.), only small amounts of soluble fibrin monomer are produced, which are not proximal to others to cause polymerization. In this case some fibrin monomer binds to a molecule of fibrinogen forming a soluble fibrin/fibrinogen dimmer. As DIC worsens in disease, the likelihood fibrin monomers to contact other monomers increases. The monomers in contact with each other bind to form soluble fibrin oligomers, of increasing length as the concentration of monomers increases in worsening disease. Oligomers consisting of greater than about eight subunits become insoluble and thrombus formation proceeds. Thus, the term ‘soluble fibrin’ refers to several moieties, including fibrin monomer, fibrin/fibrinogen dimer and fibrin oligomers. In the present application the term soluble fibrin is taken to encompass all of these soluble forms of fibrin.
Immunity and Cancer
A variety of clinical and pathologic evidence such as the presence of mononuclear cell infiltrates, composed of T-cells (Kuzushima et al., 1999), NK cells (Hokland et al., 1999), macrophages (Takanami et al., 1999) and polymorphonuclear neutrophils (PMN) (Ishikawa et al., 1986) in many tumors indicate that tumors can stimulate immune responses. Further evidence is provided by the observation of lymphocyte proliferation (hyperplasia) in lymph nodes draining sites of tumor growth (Vetto et al., 1997). In many tumors, there is evidence of cytokine effects, such as increased expression of class II major histocompatibility complex (MHC) molecules (Sikorska et al., 1999) and intercellular adhesion molecule-1 (ICAM-1; CD54) (Terol et al., 1998), suggesting an active immune response at the tumor site The spontaneous regression of tumors such as melanoma (Halliday et al., 1995) and renal cell carcinoma (Jantzer & Schendel, 1998), which are associated with dense peri- and intra-tumor lymphocytic infiltrates, is also suggestive of an immune mediated anti-tumor response.
In the blood, tumor cells encounter effector cells of the immune system, all of which are capable of anti-tumor activity, under appropriate conditions, using a variety of effector mechanisms. The most potent of these cells in the blood is considered to be the natural killer, or NK cell (Hanna & Fidler, 1980), although other cells such as T-cells (Clark et al., 1988), monocytes (Tagliabue et al., 1979) and polymorphonuclear neutrophils (Kindzelskii & Petty, 1999) show considerable anti-tumor cell activity after appropriate stimulation with cytokines or tumor cell products. Depletion of NK cells results in increased metastasis in experimental and spontaneous tumor models (Hanna, 1985). Activation of leukocytes by a variety of agents can induce increased tumoricidal activity. For example interleukin-2 (IL2) augments cytotoxicity by T-cells and NK cells (Maghazachi et al., 1988) (IL2 and IL2-activated leukocytes (LAK cells)) have been used as the basis for adoptive immunotherapy in cancer), mitogens such as phytohaemagglutinin enhance T-cell responses (Nouri et al., 1993) and bacterial lipopolysaccharide (Kildahl-Anderson & Nissen-Meyer, 1984) and interleukin-1 activate monocytes (Onozaki et al., 1985).
Most mononuclear leukocytes need to adhere to tumor cells for recognition and cytotoxicity to occur. There are a considerable number of adhesion and signaling molecules, as well as cytokines that modulate the interactions of leukocytes, platelets, tumor cells etc. with endothelium. The adhesion molecules that can mediate leukocyte cell adhesion include the leukocyte β2 integrins, LFA-1 (αLβ2; CD11a/CD18) and Mac-1 (αMβ2; CD11b/CD18) which bind to ICAM-1 (and ICAM-2) on the tumor cell (Patarroyo & Makgoba, 1989). The β2 integrins were first identified using monoclonal antibodies that inhibited lymphocyte cytotoxicity. On tumor cells which do not express ICAM-1 (many melanoma cells from fresh tumors express ICAM-1, but to very different levels (Passlick et al., 1996), other adhesion processes such as MHC class 1 and 11/T-cell receptor (Passlick et al., 1996), and the β1 (VLA) (Schadendorf et al., 1995, 1993) and β3 integrins (Liapis et al., 1997), which have also been implicated in tumor progression and metastasis, may become more prominent. In addition to adhesion, integrins also deliver stimulatory signals to cells involving tyrosine phosphorylation (Parsons, 1996) of various substrates, inositol lipid turnover (Rey-Ladino et al., 1999) and elevated levels of cytoplasmic calcium (Weismann et al., 1997). The consequences of integrin mediated signaling vary with the cell type, and include cell contraction, secretion, metabolism, cell proliferation and cell death.
Soluble Fibrin and Immunity in Cancer
Having cleaved fibrinogen, thrombin can remain bound to the formed fibrin (Hsieh, 1997) which protects it from degradation by its natural inhibitor, antithrombin. Thrombin inhibitors such as hirudin also have anti-metastatic effects (Esumi et al., 1991), possibly because thrombin can bind to tumor cells via specific receptors (Nierodzik et al., 1996) and increase their metastatic activity (Nierodzik et al., 1992). This may be necessary for post-clotting events such as activation of factor XIII, but is normally limited to the site of injury. sFn-bound thrombin is also resistant to inactivation (Hogg & Jackson, 1989). Thus, in patients with cancer, elevated levels of sFn may carry active thrombin in the circulation, perhaps explaining the relative resistance to anticoagulant therapy. Given that clotting activation seems to be important in blood-borne metastasis, high levels of circulating (procoagulant) sFn may also be a marker of poor prognosis.
Endothelial cells are critically important in tissue immunoregulation and in the process of tumor metastasis (Lafrenie et al., 1992). Leukocytes bind to endothelium using specific cell adhesion mechanisms, target to and recirculate through specific organs. This process involves three main groups of adhesive mechanism; selectins (Abbassi et al., 1993; Barkalow et al., 2000; Carlos & Harlan, 1994), integrins (Carlos & Harlan, 1994; Adams et al., 1997; Beekhuisen & Van Furth, 1993) and immunoglobulins (Carreno et al., 1995). Selectins are thought to be involved in the initial rolling of leukocytes along the endothelium, after which integrin binding occurs, followed by diapedesis into the underlying tissue. The β1 and β2 integrins are important mediators of leukocyte (and dendritic cell) adherence to CD54 and VCAM on endothelial cells. Endothelial cell CD54 expression is induced by cell activation by cytokines (Nakayama et al., 2001), thrombin (Palmblad & Lerner, 1992) and during inflammation. In addition, endothelial cells express αVβ3 (the vitronectin receptor) (Fu et al., 2001) which can also bind to fibrin(ogen) (Kubo et al., 2001). In addition to cell associated CD54, a soluble form of the molecule can be secreted by a variety of cells and significant plasma levels are observed in some diseases such as endometriosis, prostate cancer and melanoma (Becker et al., 1992; Fortis et al., 1995). This soluble CD54 has been reported to inhibit NK function in vitro (Becker et al., 1992), possibly by blocking the β2 integrin binding of the NK cells to tumor cells.
The use of activated, genetically modified dendritic cells (Agger & Hokland, 2000) as a form of adoptive immunotherapy is becoming increasingly popular. Reintroduction of these cells in adoptive immunotherapy protocols, as well as their predecessors, tumor infiltrating lymphocytes (TIL) (Aebersold et al., 1991) and lymphokine activated killer (LAK) (Fukui et al., 1988) cells requires their homing to the target tumors and crossing of the endothelium at those sites.
Inhibition of Fibrin(ogen) Binding to Cells by Specific Peptides
Fibrin(ogen) is a ligand for many biological molecules. On leukocytes it binds to several integrins, but primarily to mac1. Studies have been performed to identify the amino acid sequences on both molecules responsible for binding. Small peptides were derived from fibrinogen and mac1 and tested for their ability to inhibit fibrinogen binding. Several peptides have been identified, but the most inhibitory of them are considered to be the major binding sites.
On mac1 a sequence on the αM I-domain, 245KFGDPLGYEDVIPEADR261 (SEQ ID NO: 1; Yakubenko et al., 2001) and its complementary peptide on the fibrinogen γ-chain, 377YSMKKTTMKIIPFNRLTIG395 (SEQ ID NO: 2; Ugarova et al., 1998) have been identified. Similarly, sequences on the ICAM-1 1st Immunoglobulin domain (8KVILPRGGSVLVTC21 SEQ ID NO: 3; D'Souza et al., 1996) responsible for binding to the fibrinogen .gamma.-chain (117NQKIVNLKEKVAQLEA133 SEQ ID NO: 4; Altieri et al., 1995) are among the most potent inhibitors of fibrin(ogen) binding. ICAM-1 is expressed by many cell types including activated endothelial cells, leukocytes and many cancers. In order for fibrin(ogen) to bind to cells it must first undergo a conformational change to expose these sites which may occur when fibrinogen is immobilized on endothelial cells.
It has been demonstrated that plasma soluble fibrinogen does not adhere. Fibrin(ogen) binding to endothelial cells has been reported to enhance monocyte adherence and it is proposed that this may augment the immune response to inflammatory sites. However, no consideration is given to the elevated plasma levels of soluble fibrin (which is likely to be conformationally altered) in the blood of patients with cancer and many other conditions. In patients having elevated levels of sFn, both adherent cell types will become coated with sFn resulting in a profound inhibition of binding, characterized by an ongoing immunosuppression in these diseases, rather than the current hypothesis of enhancement of the immune response.
Soluble Fibrin in Other Diseases
In addition to cancer and metastasis, soluble fibrin is increased in a wide range of other diseases, and has been correlated with prognosis in several of them. These diseases include: nephritic syndrome (Iioka et al., 1984), diabetes mellitus (Smolenskij et al., 1979), pancreatitis (Gubergrits et al., 1993), pregnancy complications (Ostlulnd et al., 1998), coronary artery disease (Hetland et al., 2002), acute inflammation and infarction (Lindahl et al., 1990), liver disease (vanDe Water et al., 1986), myocardial infarction (Nowak et al., 1972), disseminated intravascular coagulation (DIC; Wada et al., 2003), pulmonary embolism (Bynum et al., 1976), leukemia (Zhao et al., 2000), arthritis (Koga, 2004), sepsis (Selim et al., 2005), atherosclerosis (Wang, 1996) etc. Little or no research has been performed to determine the role of soluble fibrin in the etiology of these diseases.
Thus, the prior art is deficient in an understanding the role of soluble fibrin in the etiology of cancer, cardiovascular disease, arthritis and inflammation. Additionally, there is a lack of knowledge in the prior art of therapeutic agents that can be used to treat diseases having elevated levels of soluble fibrin. The present invention fulfills this long-standing need and desire in the art.